DNA linkers

Figure 2.3 Metagenomic cloning experiments. Isolation of genomic DNA directly from environments (soil, plants, mixed environments or thermal-vent worms are the examples Illustrated here) can recover DNA fragments which could encode for enzymes. The DNA fragments can be ligated to plasmids or DNA linkers, and then subjected to functional screening (expression cloning) and/or sequence analysis. Amplification by PCR can sometimes be used to yield libraries enriched with clones containing selected sequence motifs relating to families of enzymes...

Fig. 1. The two competing classes of models for the 30-nm fiber can be distributed into (a) solenoid models and (b) crossed-linker models. For both fiber types the side and the top view are shown. Two nucleosomes that are directly connected via DNA linker are shaded in grey. In the solenoid these nucleosomes are located on the same side of the fiber requiring the linker to be bent. In the crossed-linker case they sit on opposite sides of the fiber and are connected via a straight linker.

Multiplex PCR amplification can apparently tolerate degraded DNA because the size of PCR products generated for subsequent hybridization is 100 50 base pairs. However, the 10 K mapping array and the 100 K and 500 K arrays use a specific restriction enzyme to digest the genomic DNA for subsequent ligation with a specific DNA linker. The DNA linkers then act as binding sites for the specific primer to initiate PCR amplification... 

Fig. 27 (a) Chemical structure of a polyacrylamide hydrogel, with DNA side chains providing cross-linking (b). As competing sequences are introduced, they first hybridize with a toehold overhanging sequence (c) to replace gradually the DNA linker (d), thus unbinding the network. Adapted with permission from [120]... 

Chromatin structure is organized at several levels. The basic structure of chromatin—either heterochromatin or euchromatin—is called the nucleosome. The nucleosome is a complex of 146 base pairs of DNA, wound in two turns around the outside of a disk-like complex of eight proteins (called histones). The histone core contains two copies each of four histones, H2A, H2B, H3, and H4. The histone octamer is wrapped by very close to two turns of DNA. Linker DNA and another histone (HI) join together the nucleosomes (about 65 base pairs worth). HI binds cooperatively to nucleosomes, so that a gene can be zipped up all at once by the binding of many HI molecules successively. See Figure 12-1. 

This cohesive-end method for joining DNA molecules can be made general by using a short, chemically synthesized DNA linker that can be cleaved by restriction enzymes. First, the linker is covalently joined to the ends of a DNA... 

Figure 4 Puromycin (A) is an antibiotic analog of tyrosyl tRNAthat differs by the groups in red. Once joined to a DNA linker at the 3 -end and a psoralen (B) at the 5 -end it is ready to covalently link the mRNA to nascent peptide. Photoactivation of the psoralen (green), cross-links the 5 -end of the linker and the mRNA. Once the ribosome stalls at the end of the mRNA, the puramycin enters the ribosome A-site and is transferred to the end of the newly formed protein. However, the ribosome is unable to hydrolyze the ribamine (red) amide bond thus forming a permanent link between mRNA and the encoded protein (C).

The key feature of mRNA display is that the gene (mRNA) and the encoded protein are covalently attached to each other by a puromycin-DNA linker (Figure 4). This is a key advantage over ribosome display, where the link between the mRNA and the protein is noncovalent and mediated by the ribosome. Thus mRNA display allows more stringent selection criteria to be employed that would result in the dissociation of ribosome and mRNA. 

Figure 5 Starting from natural mRNA, a cDNA library (A blue) is produced and like ribosomal display, the cDNA is transcribed into mRNA (B) with no stop codons. The 3 -end of each mRNA molecule is ligated to a short synthetic DNA linker (C) and sometimes a polyethyleneglycol spacer, which terminates with a puramycin molecule (small red sphere). The ligation is stabilized by the addition of psoralen (green clamp), which is photoactivated to covalently join both strands. Addition of crude polysomes or purified ribosomes (D) results in translation of the mRNA into protein, but the ribosome stalls at the mRNA-DNA junction. Since there are no stop codons, release factors cannot function and instead the puromycin enters the A-site of the ribosome (A). Because puramycin is an analog of tyrosyl-tRNA, the peptidyl transferase subunit catalyzes amide bond formation between the puromycin amine and the peptide carboxyl terminus, but is unable to hydrolyze the amide link (which should be an ester in tyrosyl-tRNA) to release the dimethyladenosine. The ribosome is dissociated to release the mRNA-protein fusion (E), which is protected with complementary cDNA using RT-PCR (F). The mRNA library can then be selected against an immobilized natural product probe (G), nonbinding library members washed away and the bound mRNA (H) released with SDS. PCR amplification of the cDNA provides a sublibrary (A) for another round of selection or for analysis/ sequencing.

Pairs of four different histones (H2A, H2B, H3, and H4) combine to form an eight-protein bead around which DNA is wound this bead-like structure is called a nucleosome (Figure 24-10). A nucleosome has a diameter of 10 nm and contains approximately 200 base pairs. Each nucleosome is linked to an adjacent one by a short segment of DNA (linker) and another histone (HI). The DNA in nucleosomes is further condensed by the formation of thicker structures called chromatin fibers, and ultimately DNA must be condensed to fit into the metaphase chromosome that is observed at mitosis (Figure 24-11). 

In chromatin, a stretch of"linker" DNA (080 base pairs, typically 30 base pairs) is found between adjacent chromatosomes. The complete repeating structure is called a nucleosome which contains 8 core histones + histone HI + 168 base pairs of histone-bound DNA + linker DNA. In the electron microscope, nucleosomes can be "seen" as particles arranged along the thin DNA molecule and the structure is named, after its appearance, "beads on a string." In some regions of chromatin, histone HI may be absent, leaving nucleosome core particles connected by linker DNA. 

Figure 12.1 (a) DNA-AuNPs assembled using a complementary DNA linker, (b) 13 nm... 

The strategy is based on the same organizational packing as atoms in a crystalline lattice, where particles can be designated as A, B, C, etc., based on the DNA sequence. From a thermodynamic standpoint, nanoparticles assembled through DNA linkers will... 

Figure 12.1 (a) DNA-AuNPs assembled using a complementary DNA linker, (b) 13 nm DNA-AuNPs appear red in color without linker DNA and turn blue when linker DNA induces nanopaiticle assembly, (c) Extinction spectrum of dispersed and assembled DNA-AuNPs. 

There are a number of different reasons why non-DNA linkers have been considered necessary to hold DNA molecules in tandem arrangement in chromosomes. The most relevant of these to the regulation of DNA synthesis are as follows the presumption that replication units of DNA within a sin e chromosome cannot be directly connected to one another without interruption of the DNA double helix the need for a mechanism to relieve torsion of the DNA molecule, torsion developed in connection with semiconservative replication. The latter proposal that non-DNA linkers mi t serve to allow rotation of the DNA double helix during semiconservative replication is no longer considered valid since it has been repeatedly demonstrated that a single-stranded break in a DNA double helix allows the intact chain to serve as a swivel and release torsion within the molecule (Vlnograd and Lebowitz, 1966). 

---